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CHO (Chinese Hamster Ovary) cell strain of efficiently-expressed recombinant human nerve growth factor and construction method thereof

A growth factor, high-efficiency expression technology, applied in the field of CHO cell lines of recombinant human nerve growth factor, can solve the problem of no prompt

Active Publication Date: 2012-07-18
北京福睿君安科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are no relevant reports in the existing research literature on recombinant human NGF, and the previous literature did not suggest how we can obtain a recombinant human NGF that does not require serum-free acclimatization and culture, and can be easily screened for subclones. The Way to Obtain High Efficiency Expression of Recombinant Human NGF Cell Line

Method used

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  • CHO (Chinese Hamster Ovary) cell strain of efficiently-expressed recombinant human nerve growth factor and construction method thereof
  • CHO (Chinese Hamster Ovary) cell strain of efficiently-expressed recombinant human nerve growth factor and construction method thereof
  • CHO (Chinese Hamster Ovary) cell strain of efficiently-expressed recombinant human nerve growth factor and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Construction of pOptiVEC / NGF-118 recombinant plasmid

[0052] 1. Acquisition of NGF gene

[0053] Query the sequence of human NGF protein (P01138) and gene in the UniProtKB database, and optimize the design of the NGF gene. The optimized nucleotide sequence of the NGF gene is shown in SEQ ID No.1. Jinweizhi Biotechnology Co., Ltd. Synthesized and connected to the pcDNA3.1 vector (Invitrogen Company), successfully constructed the pcDNA3.1 / NGF-118 recombinant plasmid, numbered A09025-1, and the enzyme digestion map is as follows figure 1 shown.

[0054] 2. Obtaining of pOptiVEC / NGF-118 recombinant plasmid

[0055] According to conventional molecular cloning techniques, the pcDNA3.1 / NGF-118 recombinant plasmid and pOptiVEC-polylinker vector (Invitrogen Company) were digested with Xba I and Not I (Dalian Takara Company) respectively, and then gel recovery kit (Beijing Co. Kangwei Century Biotechnology Co., Ltd.) recovered the digested NGF-118 gene fragment and...

Embodiment 2

[0056] Example 2: Obtaining the CHO DG44 cell line expressing recombinant human NGF

[0057] CHO DG44 cells are dhfr-deficient Chinese hamster ovary cell lines, purchased from Invitrogen. CHO DG44 cells were routinely passaged according to the instructions before transfection, and 3 × 10 5 Inoculate the cells / ml density in a 125ml sterile Erlenmeyer flask containing 30ml CD DG44 culture medium, so that the cell density can reach 5×10 5 Viable cells / ml, transfection steps according to Invitrogen's FreeStyle TM MAX transfection reagent instructions were carried out. Dispense 15 μl FreeStyle TM Add MAX transfection reagent and 9μg pOptiVEC / NGF-118 recombinant plasmid DNA to 1.2ml OptiCHO TM In SFM serum-free medium, mix gently and incubate at room temperature for 10 min. Slowly and evenly drop 1.2ml of the plasmid-transfection reagent mixture into the cell culture flask and place in CO 2 Incubator shaking culture. 24h after transfection, replace with CD OptiCHO containi...

Embodiment 3

[0058] Example 3: Subcloning of methylcellulose semi-solid medium to select monoclonal cell lines

[0059] CloneMatrix semi-solid medium (purchased from Genetix) was used for cloning, and the operation was performed according to the product instruction manual. Briefly, mix clonal cells into 1 × 10 3 Inoculate into 100ml CloneMatrix medium containing 400nM MTX at a density of 100ml / ml at 37°C, 5% CO 2cultured under conditions. After 14 days of culture, the independently dispersed and well-grown monoclonal cells were picked and transferred to a 96-well plate for culture. After 3 days, the culture supernatant was taken for ELISA detection. According to the results of the ELISA test, the monoclonal cells with a relatively high OD value were selected and transferred to a 24-well plate for expanded culture. After 5 days, the ELISA test was performed, and the results were as follows: image 3 shown. According to the ELISA results, 6 monoclonal wells 5, 8, 16, 20, 22 and 24 with r...

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Abstract

The intervention relates to a CHO (Chinese Hamster Ovary) cell strain of an efficiently-expressed recombinant human nerve growth factor and a construction method thereof and belongs to the field of biotechnology. The preservation number of the cell strain is CGMCC No. 4541, and the recombinant human nerve growth factor (rhNGF) with biological activity can be stably expressed. The invention also discloses a method for constructing the CHO cell strain; and through cell transfection, gene amplification and subcloning screening carried out on an amplified cell line, an efficiently-expressed monoclonal cell strain suitable for serum-free suspension culture is obtained. The invention simultaneously discloses a recombinant human nerve growth factor sequence secretly expressed by the CHO cell strain; and the secrete recombinant human nerve growth factor has good biological activity, so that PC12 multiplication can be kept outside a human body, PC12 cell differentiation is induced, and the chick embryo dorsal root ganglion nerve fiber growth is stimulated to lay a good foundation for the large-scale industrialization preparation of the recombinant human nerve growth factor.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a CHO cell strain capable of stably and efficiently secreting and expressing biologically active recombinant human nerve growth factor (rhNGF), its construction method and application. Background technique [0002] With the advancement of medicine, the survival rate of various craniocerebral trauma, acute cerebrovascular accident, spinal cord injury, neonatal ischemic and hypoxic encephalopathy, cerebral palsy and Alzheimer's disease in children continues to increase, but the neurological deficit And the poor prognosis of patients makes the medical profession face a difficult problem. At present, central nervous system injury and peripheral nerve injury have become the main diseases that threaten human health. Therefore, the development of brain neuroprotective drugs and the repair of damaged central and peripheral nerves have become a powerful driving force for the rapid developmen...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/12C12N15/85C12P21/02C12R1/91
Inventor 刘荷中乐伟史权威彭璐佳葛艳华
Owner 北京福睿君安科技有限公司
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